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Agrobacterium-mediated transformation of Eucalyptus globulus using explants with shoot apex with introduction of bacterial choline oxidase gene to enhance salt tolerance

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Abstract

Eucalyptus globulus is one of the most economically important plantation hardwoods for paper making. However, its low transformation frequency has prevented genetic engineering of this species with useful genes. We found the hypocotyl section with a shoot apex has the highest regeneration ability among another hypocotyl sections, and have developed an efficient Agrobacterium-mediated transformation method using these materials. We then introduced a salt tolerance gene, namely a bacterial choline oxidase gene (codA) with a GUS reporter gene, into E. globulus. The highest frequency of transgenic shoot regeneration from hypocotyls with shoot apex was 7.4% and the average frequency in four experiments was 4.0%, 12-fold higher than that from hypocotyls without shoot apex. Using about 10,000 explants, over 250 regenerated buds were confirmed as transformants by GUS analysis. Southern blot analysis of 100 elongated shoots confirmed successful generation of stable transformants. Accumulation of glycinebetaine was investigated in 44 selected transgenic lines, which showed 1- to 12-fold higher glycinebetaine levels than non-transgenic controls. Rooting of 16 transgenic lines was successful using a photoautotrophic method under enrichment with 1,000 ppm CO2. The transgenic whole plantlets were transplanted into potting soil and grown normally in a growth room. They showed salt tolerance to 300 mM NaCl. The points of our system are using explants with shoot apex as materials, inhibiting the elongation of the apex on the selection medium, and regenerating transgenic buds from the side opposite to the apex. This approach may also solve transformation problems in other important plants.

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Abbreviations

BA:

N6-benzyl benzyl amino purine

CaMV:

Cauliflower mosaic virus

GST:

Glutathione-S-transferase

GUS:

β-Glucuronidase

IBA:

Indole-3-butyric acid

MS:

Murashige–Skoog

NAA:

Naphthalene acetic acid

X-Gluc:

5-Bromo-4-chloro-3-indolyl-β-d-glucuronide

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Acknowledgments

This research was supported in part by the New Energy and Industrial Technology Development Organization (NEDO), Japan. We would like to thank Dr. Norio Murata for providing the codA gene and Prof. Wolfgang Schuch and Dr. Ian Jepson for providing the GST promoter.

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Authors and Affiliations

  1. Agri-Biotechnology Research Laboratory Nippon Paper Industries Co., Ltd., 5-21-1 Oji, Kita-ku, Tokyo, 114-0002, Japan

    Etsuko Matsunaga, Kazuya Nanto, Masatoshi Oishi & Teruhisa Shimada

  2. Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba, 292-0818, Japan

    Yoshihiko Morishita, Nozomu Sakurai, Hideyuki Suzuki & Daisuke Shibata

  3. R & D Division Nippon Paper Industries Co., Ltd., 5-21-1 Oji, Kita-ku, Tokyo, 114-0002, Japan

    Hiroyasu Ebinuma

Authors
  1. Etsuko Matsunaga
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  2. Kazuya Nanto
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  3. Masatoshi Oishi
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  4. Hiroyasu Ebinuma
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  5. Yoshihiko Morishita
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  6. Nozomu Sakurai
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  7. Hideyuki Suzuki
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  8. Daisuke Shibata
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  9. Teruhisa Shimada
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Corresponding author

Correspondence to Etsuko Matsunaga.

Additional information

Communicated by K. Toriyama.

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Matsunaga, E., Nanto, K., Oishi, M. et al. Agrobacterium-mediated transformation of Eucalyptus globulus using explants with shoot apex with introduction of bacterial choline oxidase gene to enhance salt tolerance. Plant Cell Rep 31, 225–235 (2012). https://doi.org/10.1007/s00299-011-1159-y

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  • DOI: https://doi.org/10.1007/s00299-011-1159-y

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